Elimination of T cells with potential autoreactivity is largely a reflection of central tolerance induced in the thymus during early T cell differentiation; this process of negative selection causes specifically-reactive T cells to undergo death in situ via apoptosis. Many aspects of central tolerance are still poorly understood, including the site of negative selection, the subsets of thymocytes involved and the role of co-stimulatory molecules. To address these questions, the susceptibility of various subsets of mature and immature T cells to negative selection will be examined under a variety of experimental conditions, both in vitro and in vivo. In vitro models will be used to define the conditions required for inducing mature and immature T cells to undergo apoptosis following short-term culture in vitro. Three forms of rapid-onset apoptosis will be examined. 1) Spontaneous apoptosis following T cell culture in medium alone, 2) TCR-mediated apoptosis induced in the absence of co-stimulation, and 3) apoptosis elicited by combined TCR ligation and co-stimulation. Particular emphasis will be placed on studying a subset of semi-mature T cells (HSA/hi CD4+ 8-cells) found in the thymic medulla. The signaling pathways involved in apoptosis and the influence of cytokines and different types of co- stimulatory molecules will be examined. Negative selection in vivo will be studied by defining which subsets of thymocytes are tolerance susceptible following injection of specific antigen into normal and TCR transgenic mice. As in vitro, the influence of cytokines an different co-stimulatory molecules on negative selection will be assessed. Information on the site of negative selection-cortex versus medulla- will be obtained by comparing tolerance induction at the level of cortical CD4+ 8- or CD4- 8+ cells. The possibility that negative selection of CD4+ 8+ cells is partly a reflection of stress following antigen injection will be examined. Resolving these questions will shed light on the basic mechanisms involved in self/non-self discrimination, the induction of autoimmune disease and the immune responses to neoplastic cells.